How many plasmids per cell

Depending on the experimental approach, the uptake of two or even many different plasmids may be required, whereas in other cases uptake of multiple plasmids should be avoided. If a population of cells which is transfected by a library expressing different sequences e.

For other experimental purposes however, experimental conditions have to be adjusted so that most cells of a population take up not only one but two or even many different plasmids.

A fascinating example of multiple plasmid uptake is the reconstruction of the B-cell receptor signaling pathway in Drosophila Schneider cells, where many plasmids encoding different protein species are simultaneously expressed in the same cell and the kinase activity depending on the introduced components is assayed biochemically [ 2 ].

Another application requiring multiple plasmid uptake is the generation of stable transgenic lines by gene replacement taking advantage of an artificial, Cre-recombinase catalyzed, loxP -mediated homologous recombination, allowing the insertion of different transgenes at reproducible sites into a eukaryotic genome with high efficiency and yield [ 3 ].

This is accomplished by co-transfection of one plasmid carrying the transgene flanked by loxP and loxP sites together with a second plasmid expressing bacterial Cre-recombinase.

Another type of experiment requiring multiple plasmid uptake is the simultaneous expression of two RNAi constructs in one cell or the simultaneous expression of a sense and a corresponding antisense construct as it is reported for Physarum polycephalum in the present paper.

In this work, we first estimate the concentration-dependence of the number of plasmids Jurkat cells take up during electroporation and then apply the principle to show and quantify the effectiveness of antisense expression in P. Fluorescence micrographs were taken with a Zeiss Axiocam color CCD camera and processed using the Axiovision software package.

Since it did not complement the promoter proximal Not I site, this site was destroyed, while the one distal to the promoter was maintained. The Escherichia coli trxA gene was ligated into MCS2 in order to allow display of peptide aptamers in future studies, using the introduced restriction sites. Physarum flagellates strain WT31 were prepared as described [ 6 ]. On the 6th day after inoculation the plate was flooded with 10 ml of water and incubated for 1—2 h.

During that time, developing cysts transform into flagellates. When cultures became older than 6 days, the hatching efficiency continually decreased along with progressing cyst differentiation and therefore such cultures were not used in the experiments.

The supernatant containing the flagellates was carefully collected from the tilted plate using a 5-ml cotton-plugged glass pipette. Care was taken not to carry over any untransformed cysts, sitting on the agar surface, bacterial debris or any agar material.

After the incubation step, cells were pelleted as described above, resuspended in 2 ml of deionized water and evaluated with the fluorescent microscope. Axenically grown LU cells qualitatively yielded identical results i. Under experimental conditions that are commonly used to transfect cells with DNA, the number of plasmid molecules added to the sample usually is in great excess compared to the number of cells present and the probability that a given plasmid molecule is taken up by a cell is extremely low.

In consequence, the probability that a given cell will take up at least one DNA molecule depends on the average number of molecules taken up which in turn is proportional to the DNA concentration in the sample.

Based on the equation describing a Poisson distribution, a dose-effect curve can be calculated displaying the probability that one or more DNA molecules are taken up by a cell as a function of the mean number of DNA molecules taken up on average see Appendix A for details.

This average number is proportional to the concentration of DNA in the sample. If the probability of taking up one or more molecules i. This way of curve fitting has been employed for example for the evaluation of dose—response curves in photo-regulated biological processes [ 10 ]. If all individual cells of a population would behave identical regarding their ability to take up a molecule of plasmid DNA, then at a given DNA concentration the percentage of cells that take up none, one, two or more plasmid molecules could be calculated directly from the dose-effect curve.

However, under true experimental conditions cells do not necessarily behave identical and might not be equally susceptible to DNA uptake. Assume that during electroporation there are some cells that have many pores open for DNA uptake, others only one or two and other cells have no pores at all.

Then, at non-saturating DNA concentration the probability that at least one DNA molecule enters a cell is higher for a cell which has several pores open for DNA uptake compared to a cell that has only one or no pore at all. The opposite, i. A simple solution to this problem is the transfection with a mixture of two plasmids that are identical except for a few point mutations e. If at varying total DNA concentration the ratio of EYFP and ECFP encoding DNA is kept constant and cells that are double fluorescent are related to the number of cells which show only one of the two fluorescent proteins, it can be analyzed how precisely the Poisson distribution is met.

If the number of cells that express two fluorescent proteins follows a Poisson distribution, this demonstrates that the plasmid concentration-dependent change in the number of fluorescent cells reflects the stochastics of single molecule uptake. Such an analysis indicates how homogeneous a cell population behaves with respect to its ability to be transformed with DNA. Schematic representation of the experiment for determining the number of single and double transfected Jurkat cells by random uptake from a mixture of two plasmids.

Both plasmids only differ in the few mutations that determine the excitation wavelength maximum blue or yellow of the respective fluorescent protein. Cells were transiently transfected by electroporation and after incubation for one day — to allow for sufficient protein expression — the fluorescence of the transfected cells was scored by fluorescence microscopy using appropriate filter sets to distinguish the two proteins.

The occurrence of the three cell types depended on the absolute concentration of the two plasmids in the plasmid mix used for transfection Fig. Arrows indicate cells that are single fluorescent. Dose-effect curves of Jurkat double transfection experiments. Each symbol represents the result of an independent experiment. Error bars indicate the estimated counting error for one exemplary experiment. The deviation of the data points marked by open circles from the calculated curve suggests that the culture used in this experiment was less homogeneous than the cultures used in the experiments represented by the other symbols.

The finding that at a given concentration of pSM-EYFP and pSM-ECFP double fluorescent cells simultaneously expressing both proteins could be observed side by side with single fluorescent cells expressing only one of the two proteins in one sample suggested that fluorescent cells have taken up only one, two or a few plasmid molecules.

To quantify this effect, the concentration of the two plasmids was altered while their concentration relative to each other was kept constant.

To correct for possible changes in the overall efficiency of transient transfection, the number of double fluorescent cells relative to the total number of single fluorescent cells was determined by counting. By plotting the relative frequency of double fluorescent cells versus the concentration of either of the plasmids, a dose-effect curve was obtained Fig.

Fitting the experimental data to a dose-effect curve calculated from the Poisson distribution assuming that one plasmid molecule is sufficient to make a cell fluorescent, gave a reasonably good result, but did not rule out the possibility that the true curve might be less steep than the calculated one.

This phenomenon would occur if the susceptibility of individual Jurkat cells within a population would vary to a certain extent. To account for this possible effect, the fitting routine described earlier [ 11 ] was modified in that it assumed two populations of cells of different susceptibility that occurred at a relative frequency of one to one which would be the worse case; see Eq.

Following this assumption, the data could be fitted equally well by assuming a homogeneous population or by assuming an inhomogeneous population; however, only when the susceptibility of the two populations differed by a factor of three or less Fig. In other words, a certain fraction of double fluorescent cells might have contained more plasmid molecules than predicted by the Poisson distribution of a homogeneous population. Irrespective of this quantitative argument, in any case the qualitative argument holds that we observe the activity caused by one or two to few plasmid molecules per cell, since single fluorescent cells along with double fluorescent cells were observed in all experiments performed.

The experimental and computational results allow three conclusions to be drawn: 1 The plasmid concentration at which most of the cells definitely take up only one plasmid molecule can be determined. Cloning of vectors was performed using E. Induction experiments were carried out in minimal medium composed of 0. The construction of vectors was carried out according to standard protocols.

For all constructs, positive clones were identified by colony PCR and verified by plasmid isolation, restriction digestion and sequencing of the insert using the proposed SEVA standard primers for T0 and T1 terminators [ 1 ]. Plasmids were transferred into E. The alignment of the instrument with fluorescent beads and the sheath buffer composition here using a twofold dilution are given in [ 41 ].

Detailed statistics on flow cytometry samples can be found in Additional file 1. Data acquisition and cell sorting was performed as described in [ 7 ]. The most accurate sorting mode single cell and one drop mode was used for highest purity. As a control for accurate sorting, a cell sample spiked with beads was used to sort beads per well as the no-template-control. DNA was extracted from whole sorted cells by the heat treatment method described in [ 7 ]. Different incubation times for heat treatment ranging from 0 to 60 min were tested with ddPCR and the condition with the highest obtained concentration for genomic DNA 10 min was chosen for further experiments Additional file 3 : Figure S4.

Probes and primers were tested according to the dMIQE guidelines [ 45 ], including optimal concentration, annealing temperature, formation of a single product using qRT-PCR and gel electrophoresis , and discrimination of negative and positive droplets in ddPCR. Droplet digital PCR was performed as described in [ 7 ]. Briefly, a duplex reaction set-up was used with simultaneous detection of a reference gene and a target gene.

A master mix containing all ingredients except the template was prepared and added to the heat-treated samples in 8-well strips. Generated droplets were transferred to a twin. Induction experiments were performed with two independent biological replicates, and all PCR experiments were performed with four technical replicates per condition at the stage of cell sorting. Data were exported as text file and further analyzed using R v3.

Outliers were not removed except for known pipetting errors. Nucleic Acids Res. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. Google Scholar. Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria.

Metab Eng. Summers DK. The kinetics of plasmid loss. Trends Biotechnol. Non-random distribution of macromolecules as driving forces for phenotypic variation. Curr Opin Microbiol. Subpopulation-proteomics in prokaryotic populations. Curr Opin Biotechnol. Anal Chem. Rapid optimization of gene dosage in E. J Biol Eng. Standards not that standard. Article Google Scholar. Engineering BioBrick vectors from BioBrick parts.

SEVA, 2. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Mol Microbiol. Determination of plasmid copy number by fluorescence densitometry.

Ryan W, Parulekar SJ. Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM harboring a high copy number plasmid. Biotechnol Bioeng. The host range of RK2 minimal replicon copy-up mutants is limited by species-specific differences in the maximum tolerable copy number. Quantitative real-time polymerase chain reaction for determination of plasmid copy number in bacteria.

J Microbiol Methods. Time-course determination of plasmid content in eukaryotic and prokaryotic cells using real-time PCR. Mol Biotechnol. Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes. Microb Cell Fact.

Variability in subpopulation formation propagates into biocatalytic variability of engineered Pseudomonas putida strains.

Front Microbiol. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol. Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Mol Biol. A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli.

Bistability, epigenetics, and bet-hedging in bacteria. Annu Rev Microbiol. Polar fixation of plasmids during recombinant protein production in Bacillus megaterium results in population heterogeneity.

Appl Environ Microbiol. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.

Schmidt L, Inselburg J. ColE1 copy number mutants. CAS Google Scholar. ColE1-plasmid production in Escherichia coli : mathematical simulation and experimental validation. Front Bioeng Biotechnol. Camps M. Modulation of ColE1-like plasmid replication for recombinant gene expression. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations. J Mol Biol. Control of tryptophan synthetase amplified by varying the numbers of composite plasmids in Escherichia coli cells.

Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production. Klumpp S. Growth-rate dependence reveals design principles of plasmid copy number control. A reduced genome decreases the host carrying capacity for foreign DNA. Bacterial fitness and plasmid loss: the importance of culture conditions and plasmid size.

Can J Microbiol. Transferable colicinogenic factors as mobilizing agents for extrachromosomal streptomycin resistance. Multicopy plasmids are clustered and localized in Escherichia coli. Comparison of preservation methods for bacterial cells in cytomics and proteomics. J Integr Omics. Cytometric fingerprinting for analyzing microbial intracommunity structure variation and identifying subcommunity function.

Nat Protoc. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. Marshall OJ. PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. BMC Bioinform. Clin Chem.

DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials. Anal Bioanal Chem. Download references. MJ designed the study, performed vector cloning, cultivation experiments, cell sorting and PCN determination and wrote the manuscript, CV performed vector cloning, cultivation experiments and PCN determination, TH carried out flow cytometry and cell sorting, HH and SM wrote the manuscript.

A simple way to think about this us to imagine a plasmid with a copy number of two see figure 1A. The regulatory machinary of the origin of replication is set to keep the copy number at two, but in a double transformant containing two different plasmid clones with the same origin of replication the copy number of the replicon is already two, so no replication will occur. The two plasmids will then be seggregated to different daughter cells after cell division, so each daughter cell will contain a homogenous plasmid population.

This is all very well, but if the cells are plated before they have a chance to divide, this could —to my mind— result in a clone containing two different plasmid clones. Moreover, most plasmids have a copy number higher than 2, so the situation is more complicated. Even with a copy number of 4 the chances of propigating a heterogenous plasmid population is increased, it would seem.

The commonly understood wisdom is that the two plasmid clones will have small differences that cause one to have a faster replication rate, or increased toxicity, over the other.

This is said to cause the plasmids to be replicated assymetrically, contributing to the eventual loss of one of the clones. This is apparently true in many cases, but a paper from Velappan et al [3] reports that in double transformants containing two plasmids with identical origins of replication can be stably maintained together in the same E.

If you take this is the context of a transformation, to me this seems like it is entirely possible that Beheroze has obtained a colony from that contains a mixed plasmid population. This could have arisen from a single E. From this it would seem that in any experiment where a mixed plasmid population is being transformed and a homogenous cloned is desired, double transformations should be borne in mind, diagnosed and avoided where possible.

Use low concentrations of plasmid. Goldsmith et al showed that double transformation increased exponentially with plasmid concentration, so keeping the plasmid concentration as low as possible, given the constraints of your experiment, should help. Check clones using colony PCR or sequencing. If you have a double transformant it will give two bands.

Beheroze has identified double clones by sequencing the resulting plasmid preps and noticing that the mixture might contain two different sequences. Choose more than one clone. If you do have some double transformants, they should be in a minority if you choose a bank of positive clones. The singly-transformed positives should be easily indentified by colony PCR or sequencing.

Positive clones should be isolated and used to innoculate a fresh culture, plated at low density on an agar plate and individual colonies re-analysed. Alternatively, you could isolate the plasmid s from the positive clone and re-transform. If your plasmid population was mixed, you will get a plate of mostly single transformants containing one or other of the originally co-transformed plasmid.